Expression and localization of Cyclin D1/Nanog and NF-κB/Bax protein in dysplastic testicles of mice.

Testicular dysplasia significantly impairs male reproductive capacity. This study investigated the expression of Cyclin D1/Nanog and NF-κB/Bax in dysplastic testes of mice using histological staining, Western blotting, and immunohistochemistry. The results showed that Nanog and Bax expression were significantly higher in dysplastic testicular tissue than in normal tissue (P < 0.01). Cyclin D1 protein expression was higher in normal testis tissue than in dysplastic testis (P < 0.01). NF-κB was highly expressed in cryptorchid and normal testis with no significant difference (P > 0.05). Immunolocalization revealed that Nanog, NF-κB, and Bax were expressed in the cytoplasm of Leydig and spermatogenic cells. Cyclin D1 primarily expressed in the nucleus of Sertoli cells. These findings suggest that altered expression of Nanog, Cyclin D1, and Bax may contribute to testicular dysplasia. This study provides a scientific foundation for detecting testicular dysplasia and selecting appropriate animal models, ultimately informing strategies to improve male reproductive health.

Influenza A virus infection activates caspase-8 to enhance innate antiviral immunity by cleaving CYLD and blocking TAK1 and RIG-I deubiquitination.

Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.

Influenza A virus infection activates STAT3 to enhance SREBP2 expression, cholesterol biosynthesis, and virus replication.

Cellular cholesterol plays an important role in influenza A virus (IAV) endocytosis and replication. However, how IAV infection regulates cholesterol biosynthesis remains poorly understood. Here, we report that IAV infection activates SREBP2 and induces the expression of HMGCR, a rate-limiting enzyme in cholesterol synthesis pathway. SREBP2 deficiency suppresses IAV-induced HMGCR expression and virus replication. Mechanistically, IAV infection activates JAK2 and STAT3, inhibition of JAK2 and STAT3 activity by their inhibitors or by gene knockout downregulates IAV-induced SREBP2 and HMGCR expression and IAV replication, reduces the content of cellular cholesterol and virus binding to host cells. Exogenous cholesterol reverses the inhibitory effect of S3I-201 and STAT3 deficiency on virus replication. STAT3 or JAK2 overexpression increases the expression of SREBP2 and its downstream target genes, leading to increased IAV replication. These observations collectively suggest that STAT3 activation facilitates IAV replication by inducing SREBP2 expression and increasing cholesterol biosynthesis.

Influenza virus infection activates TAK1 to suppress RIPK3-independent apoptosis and RIPK1-dependent necroptosis.

Many DNA viruses develop various strategies to inhibit cell death to facilitate their replication. However, whether influenza A virus (IAV), a fast-replicating RNA virus, attenuates cell death remains unknown. Here, we report that IAV infection induces TAK1 phosphorylation in a murine alveolar epithelial cell line (LET1) and a murine fibroblastoma cell line (L929). The TAK1-specific inhibitor 5Z-7-Oxzeneonal (5Z) and TAK1 knockout significantly enhance IAV-induced apoptosis, as evidenced by increased PARP, caspase-8, and caspase-3 cleavage. TAK1 inhibition also increases necroptosis as evidenced by increased RIPK1S166, RIPK3T231/S232, and MLKLS345 phosphorylation. Mechanistically, TAK1 activates IKK, which phosphorylates RIPK1S25 and inhibits its activation. TAK1 also activates p38 and its downstream kinase MK2, which phosphorylates RIPK1S321 but does not affect RIPK1 activation. Further investigation revealed that the RIPK1 inhibitor Nec-1 and RIPK1 knockout abrogate IAV-induced apoptosis and necroptosis; re-expression of wild-type but not kinase-dead (KD)-RIPK1 restores IAV-induced cell death. ZBP1 knockout abrogates IAV-induced cell death, whereas RIPK3 knockout inhibits IAV-induced necroptosis but not apoptosis. 5Z treatment enhances IAV-induced cell death and slightly reduces the inflammatory response in the lungs of H1N1 virus-infected mice and prolongs the survival of IAV-infected mice. Our study provides evidence that IAV activates TAK1 to suppress RIPK1-dependent apoptosis and necroptosis, and that RIPK3 is required for IAV-induced necroptosis but not apoptosis in epithelial cells.

Nonstructural Protein A238L of the African Swine Fever Virus (ASFV) Enhances Antiviral Immune Responses by Activating the TBK1-IRF3 Pathway.

African swine fever virus (ASFV) is a double-stranded DNA virus with an envelope. ASFV has almost the largest genome among all DNA viruses, and its mechanisms of immune evasion are complex. Better understanding of the molecular mechanisms of ASFV genes will improve vaccine design. A238L, a nonstructural protein of ASFV, inhibits NF-κB activation by suppressing the HAT activity of p300. Whether A238L also affects the transcriptional activity of IRF3 remains unexplored. Here we first confirmed the ability of A238L to suppress NF-κB-activity in L929 cells. A238L inhibits the expression of proinflammatory cytokine genes. In contrast, A238L increased the phosphorylation levels of TBK1 and IRF3 in three different cell lines. A238L increases the IRF3-driven promoter activity and induces IRF3 nuclear translocation. Furthermore, A238L enhanced innate antiviral immunity in the absence or presence of poly d (A:T) or poly (I:C) stimulation, or herpes simplex virus type 1 (HSV-1) or Sendai virus (SeV) infection. This study reveals a previously unrecognized role of A238L in promoting antiviral immune responses by TBK1-IRF3 pathway activation.

Special Staining and Protein Expression of VEGF/EGFR and P53/NF-κB in Cryptorchid Tissue of Erhualian Pigs.

Erhualian pigs exhibit one of the highest reproductive rates globally, and cryptorchidism is a crucial factor affecting reproductive abilities of boars. This investigation focused on cryptorchid tissues from Erhualian pigs, where the histological structure of cryptorchidism was observed using specialized staining. In addition, protein expression of P53/NF-κB in cryptorchid tissues was assessed using Western blot and immunohistochemistry. In comparison to normal Erhualian testes, Masson's trichrome staining indicated a reduction in collagen fibers in the connective tissue and around the basal membrane of the seminiferous tubules in cryptorchid testes. Moreover, collagen fiber distribution was observed to be disordered. Verhoeff Van Gieson (EVG) and argyrophilic staining demonstrated brownish-black granular nucleoli organized regions in mesenchymal cells and germ cells. When compared to normal testicles, the convoluted seminiferous tubules of cryptorchids exhibited a significantly reduced number and diameter (p < 0.01). Notably, VEGF/EGFR and P53/NF-κB expression in cryptorchidism significantly differed from that in normal testes. In particular, the expression of VEGF and P53 in cryptorchid tissues was significantly higher than that in normal testes tissues, whereas the expression of EGFR in cryptorchid tissues was significantly lower than that in normal testes tissues (all p < 0.01). NF-κB expressed no difference in both conditions. The expressions of VEGF and NF-κB were observed in the cytoplasm of testicular Leydig cells and spermatogenic cells, but they were weak in the nucleus. EGFR and P53 were more positively expressed in the cytoplasm of these cells, with no positive expression in the nucleus. Conclusion: There were changes in the tissue morphology and structure of the cryptorchid testis, coupled with abnormally high expression of VEGF and P53 proteins in Erhualian pigs. We speculate that this may be an important limiting factor to fecundity during cryptorchidism.

Vemurafenib activates the sonic hedgehog pathway and promotes thyroid cancer stem cell self-renewal.

B-Raf kinase inhibitors such as vemurafenib (PLX4032) and dabrafenib have limited therapeutic efficacy on BRAF-mutated thyroid cancer. Cancer stem cells (CSCs) play important roles in tumor recurrence, drug resistance, and metastasis. Whether CSCs play a role in dampening the antitumor activity of B-Raf kinase inhibitors remains unknown. Here, we report that vemurafenib (PLX4032) induced the expression of several stemness-related genes including Gli1, Snail, BMI1, and SOX2 in two anaplastic thyroid cancer cell lines, SW1736 and 8505C, but decreased the expression of these genes in A375 cells, a human melanoma cell line. PLX4032 promoted thyroid cancer stem cell self-renewal, as evidenced by increased numbers of aldehyde dehydrogenase-positive cells and thyrospheres. Mechanistically, PLX4032 activates the PI-3 and mitogen-activated protein kinase pathways through HER3 to cross-activate Gli1, a transcription factor of the sonic hedgehog (Shh) pathway. GANT61, a specific inhibitor of Gli1, blocked the expression of the stemness-related genes in PLX4032-treated thyroid cancer cells in vitro and in vivo in two thyroid cancer xenograft models. GANT61 treatment alone weakly inhibited SW1736 tumor growth but enhanced the antitumor activity of PLX4032 when used in combination. Our study provides mechanistic insights into how thyroid cancer poorly responds to B-Raf kinase inhibitors and suggests that targeting B-Raf and the Shh pathway in combination may overcome thyroid cancer drug resistance.

Xanthine oxidoreductase mediates genotoxic drug-induced autophagy and apoptosis resistance by uric acid accumulation and TGF-ß-activated kinase 1 (TAK1) activation.

Autophagy is a highly conserved cellular process that profoundly impacts the efficacy of genotoxic chemotherapeutic drugs. TGF-ß-activated kinase 1 (TAK1) is a serine/threonine kinase that activates several signaling pathways involved in inducing autophagy and suppressing cell death. Xanthine oxidoreductase (XOR) is a rate-limiting enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid and hydrogen peroxide in the purine catabolism pathway. Recent studies showed that uric acid can bind to TAK1 and prolong its activation. We hypothesized that genotoxic drugs may induce autophagy and apoptosis resistance by activating TAK1 through XOR-generated uric acid. Here, we report that gemcitabine and 5-fluorouracil (5-FU), two genotoxic drugs, induced autophagy in HeLa and HT-29 cells by activating TAK1 and its two downstream kinases, AMP-activated kinase (AMPK) and c-Jun terminal kinase (JNK). XOR knockdown and the XOR inhibitor allopurinol blocked gemcitabine-induced TAK1, JNK, AMPK, and Unc51-like kinase 1 (ULK1)S555 phosphorylation and gemcitabine-induced autophagy. Inhibition of the ATM-Chk pathway, which inhibits genotoxic drug-induced uric acid production, blocked gemcitabine-induced autophagy by inhibiting TAK1 activation. Exogenous uric acid in its salt form, monosodium urate (MSU), induced autophagy by activating TAK1 and its downstream kinases JNK and AMPK. Gene knockdown or the inhibitors of these kinases blocked gemcitabine- and MSU-induced autophagy. Inhibition of autophagy by allopurinol, chloroquine, and 5Z-7-oxozeaenol (5Z), a TAK1-specific inhibitor, enhanced gemcitabine-induced apoptosis. Our study uncovers a previously unrecognized role of XOR in regulating genotoxic drug-induced autophagy and apoptosis and has implications for designing novel therapeutic strategies for cancer treatment.

Expression Analysis of Molecular Chaperones Hsp70 and Hsp90 on Development and Metabolism of Different Organs and Testis in Cattle (Cattle-yak and Yak).

Hsp70 and Hsp90 play an important role in testis development and spermatogenesis regulation, but the exact connection between Hsp70 and Hsp90 and metabolic stress in cattle is unclear. Here, we focused on the male cattle−yak and yak, investigated the expression and localization of Hsp70 and Hsp90 in their tissues, and explored the influence of these factors on development and metabolism. In our study, a total of 54 cattle (24 cattle−yaks and 30 yaks; aged 1 day to 10 years) were examined. The Hsp90 mRNA of the cattle−yak was first cloned and compared with that of the yak, and variation in the amino acid sequence was found, which led to differences in protein spatial structure. Using real-time quantitative PCR (RT-qPCR) and Western blot (WB) techniques, we investigated whether the expression of Hsp70 and Hsp90 mRNA and protein are different in the cattle−yak and yak. We found a disparity in Hsp70 and Hsp90 mRNA and protein expression in different non-reproductive organs and in testicular tissues at different stages of development, while high expression was observed in the testes of both juveniles and adults. Moreover, it was intriguing to observe that Hsp70 expression was significantly high in the yak, whereas Hsp90 was high in the cattle−yak (p < 0.01). We also examined the location of Hsp70 and Hsp90 in the testis by immunohistochemical (IHC) and immunofluorescence (IF) techniques, and the results showed that Hsp70 and Hsp90 were positive in the epithelial cells, spermatogenic cells, and mesenchymal cells. In summary, our study proved that Hsp70 and Hsp90 expressions were different in different tissues (kidney, heart, cerebellum, liver, lung, spleen, and testis), and Hsp90 expression was high in the testis of the cattle−yak, suggesting that dysplasia of the cattle−yak may correlate with an over-metabolism of Hsp90.

Development of a Duplex Insulated Isothermal PCR Assay for Rapid On-Site Detection and Differentiation of Genotypes 1 and 2 of African Swine Fever Virus.

Genotype II African swine fever virus (ASFV) has been plaguing Asian pig industry since 2018. Recently, genotype I ASFV was reported for the first time in China. Since there is no commercial vaccine available against ASFV, early onsite detection and quick culling procedures are commonly used by many countries all over the world. It is important that the above two genotypes of ASFV could be quickly differentiated during onsite detection at the same time. In this study, we established a sensitive and simple Fluorescent Probe Hydrolysis-Insulated isothermal PCR (iiPCR) that can detect and differentiate two genotypes of ASFV within 40 minutes. The positive or negative results of tested samples were displayed on the screen of the device automatically after PCR amplification was complete. The detection limit of the iiPCR was tested to be 20 copies for both genotype I and genotype II ASFVs. There was no cross-reactivity with other swine viruses by using the established iiPCR. Fifty-eight ASFV positive samples confirmed by National ASF Reference Laboratory were subjected to the established duplex iiPCR for genotype differentiation. The results showed that all these ASFV-positive samples belong to genotype II. At last, we found serum samples could be directly used as the templates for iiPCR without comprising sensitivity and specificity. Therefore, the duplex iiPCR established in study provide a useful tool for ASFV onsite detection and genotype differentiation.

Expression of tumour transcription factor GLI1 in canine mammary tumours tissue.

BACKGROUND: Mammary tumor is one of the most common diseases of canine in pet clinics. OBJECTIVES: This study investigates the distribution and expression of the tumor transcription factor GLI1 and the downstream proteins, Bmi1 and Sox2, in canine mammary tumors and paracancerous tissues. METHODS: Cancerous and paracancerous normal mammary tissues were detected using western blotting (WB), and immunohistochemistry. RESULTS: The results showed that the histopathology of different types in mammary tumors by microscopic observation. GLI1/Bmi1/Sox2 expression was significantly higher in canine mammary invasive carcinoma than in ductal carcinoma and adjacent normal mammary tissues (p < 0.01). The expression of GLI1 in invasive carcinoma tissues was significantly higher than Bmi1 and Sox2, while Sox2 expression in ductal carcinoma tissues was significantly higher than GLI1 and Bmi1 (p < 0.01). GLI1/Bmi1/Sox2 all showed positive reactions in both mammary tumor and adjacent normal mammary tissues with immunohistochemistry. GLI1 and Sox2 showed strong positive staining in the cytoplasm of invasive mammary carcinoma and ductal carcinoma cells, and weak positive staining in the nuclei. The positive Bmi1 reaction was mainly concentrated in the cytoplasm of invasive carcinoma and ductal carcinoma cells, while the positive reaction on the cell membrane was weak. CONCLUSIONS: We speculate that GLI1 and related proteins play an important role in regulating the proliferation and differentiation of tumors. Therefore, it provides important reference for the pathogenesis and pathogenicity of canine mammary tumor.

Effects of testosterone on skin structure and factors related to androgen conversion and binding in Hetian sheep.

The effects of androgens on human skin mainly include the regulation of growth and differentiation of hair follicles and sebaceous glands. Androgens may have some physiological roles in sheep skin that are similar to those of humans, but further confirmation is needed. Therefore, Hetian sheep were chosen in this study as an animal model to explore the effects of testosterone on skin structure and factors related to androgen conversion and binding in Hetian sheep. The sheep were treated with different concentrations of testosterone for 42 days. Skin tissue sections were prepared and then subjected to hematoxylin-eosin, Sacpic, Masson's trichrome, and Oil Red O staining to observe changes in skin morphology. Changes in the content of blood-related factors were also detected using ELISA kits. The skin tissue distribution of androgen receptor was explored by immunohistochemistry and immunofluorescence assays. The results showed that testosterone significantly increases the sebaceous gland area and stimulates the formation of new sebaceous glands. Further exploration revealed that testosterone promotes the proliferation of sebaceous gland juvenile cells. However, testosterone was found to have no significant effect on hair follicle density and hair follicle structure. Testosterone increased dihydrotestosterone levels but decreased 5α-reductase 1 and 5α-reductase 2 levels. The androgen receptors were distributed in the hair follicles, sebaceous glands, and some major skin appendages of Hetian sheep. This study suggests that androgens can be effective in regulating sebum production in sheep. This study will help advance research efforts to further explore the molecular and cellular mechanisms by which androgens modify sheep follicles and sebaceous glands.

Identification of key genes and signaling pathways related to Hetian sheep wool density by RNA-seq technology.

Hetian sheep is a breed of sheep unique to the Hetian area of Xinjiang whose wool is used for producing blankets. Individual differences and hair follicle density are the key factors affecting wool production. Therefore, this study aimed to assess the Hetian sheep having different wool densities to statistically analyze the wool traits and hair follicle parameters. Furthermore, the transcriptome sequencing analysis was performed on the skins with different wool densities. The results showed that wool quantity and total hair follicle density of the high wool density sheep was significantly higher than low wool density sheep. The sheepskin with high wool density was found to grow more and finer wool than sheepskin with low wool density. A total of 1,452 differentially expressed genes were screened from the two sets of samples, including 754 upregulated and 698 downregulated genes. The differentially expressed genes were involved in the TGF-ß/BMP and MAPK signaling pathways related to hair growth. Eleven differentially expressed genes belonging to the KAPs and KIFs might affect the fineness of the wool. The key genes, like the TNF, MAP2K2, INHBA, FST, PTPN11, MAP3K7, KIT, and BMPR1A, were found to probably affect the growth and density of the wool. The qPCR verified eight genes related to the MAPK pathway whose gene expression trends were consistent with the transcriptome sequencing results. This study furnishes valuable resources for enhancing the quality and production of wool in the Hetian sheep.

Uric acid accumulation in DNA-damaged tumor cells induces NKG2D ligand expression and antitumor immunity by activating TGF-ß-activated kinase 1.

DNA damage by genotoxic drugs such as gemcitabine and 5-fluorouracil (5-FU) activates the ataxia telangiectasia, mutated (ATM)-Chk pathway and induces the expression of NKG2D ligands such as the MHC class I-related chain A and B (MICA/B). The mechanisms underlying this remain incompletely understood. Here we report that xanthine oxidoreductase (XOR), a rate-limiting enzyme that produces uric acid in the purine catabolism pathway, promotes DNA damage-induced MICA/B expression. Inhibition of the ATM-Chk pathway blocks genotoxic drug-induced uric acid production, TGF-ß-activated kinase 1 (TAK1) activation, ERK phosphorylation, and MICA/B expression. Inhibition of uric acid production by the XOR inhibitor allopurinol blocks DNA damage-induced TAK1 activation and MICA/B expression in genotoxic drug-treated cells. Exogenous uric acid activates TAK1, NF-κB, and the MAP kinase pathway. TAK1 inhibition blocks gemcitabine- and uric acid-induced MAP kinase activation and MICA/B expression. Exogenous uric acid in its salt form, monosodium urate (MSU), induces MICA/B expression and sensitizes tumor cells to NK cell killing. MSU immunization with irradiated murine breast cancer cell line RCAS-Neu retards breast cancer growth in syngeneic breast cancer models and delays breast cancer development in a somatic breast cancer model. Our study suggests that uric acid accumulation plays an important role in activating TAK1, inducing DNA damage-induced MICA/B expression, and enhancing antitumor immunity.

Development and application of a duplex real-time PCR assay for differentiation of genotypes I and II African swine fever viruses.

Genotype II African swine fever virus (ASFV) has been plaguing Chinese pig industry and caused severe morbidity and mortality of pigs resulting in huge economic losses since its first report in August 2018. Most recently, two genotype I ASFVs with low virulence but efficient transmissibility in pigs were reported in China, which makes the diagnosis and control of this lethal disease more challenging. Therefore, it is prerequisite and important to differentiate genotype I from genotype II upon ASFV outbreaks before making any stringent control procedures. In this study, a duplex real-time PCR assay based on ASFV E296R gene was established which could simultaneously detect genotypes I and II ASFVs with two pairs of primers and two probes. Plasmid containing ASFV genes was used to test the sensitivity, repeatability, and reproducibility. DNA or cDNA samples of ASFV and other swine viruses were used to test the specificity. The results showed that the established duplex real-time PCR assay has satisfied specificity, sensitivity, repeatability, and reproducibility. In addition, the assay was applied to differentiate 84 ASFV positive clinical samples including lymph nodes, spleen, kidney, lung, liver, blood, nasal swab, and environmental swab samples which were sent to National ASF Reference Laboratory from April 2020 to September 2021. The results showed that all these ASFV positive samples belong to genotype II ASFV. The established duplex real-time PCR in this study provides a powerful tool for rapid detection and differentiation between genotypes I and II ASFVs and will facilitate efficient control of ASFV in China.

Nanofiber-mediated sequential photothermal antibacteria and macrophage polarization for healing MRSA-infected diabetic wounds.

BACKGROUND: Diabetic wound healing remains a challenge because of its susceptibility to drug-resistant bacterial infection and its persistent proinflammatory state. Switching from proinflammatory M1 macrophages (Mφs) to proregenerative M2 dominant Mφs in a timely manner accelerates wound healing by coordinating inflammatory, proliferative, and angiogenic processes. METHODS: We propose a sequential photothermal antibacterial and subsequent M2 Mφ polarization strategy based on nanofibers (NFs) consisting of polydopamine (PDA) coating on curcumin (Cur) nanocrystals to treat Methicillin-resistant Staphylococcus aureus (MRSA)-infected diabetic wounds. RESULTS: The PDA/Cur NFs showed excellent photothermal conversion and antibacterial effects due to the PDA shell under laser irradiation, consequently resulting in the release of the inner Cur with the ability to promote cell proliferation and reinforce the M2 Mφ phenotype in vitro. In vivo studies on MRSA-infected diabetic wounds showed that PDA/Cur NFs not only inhibited MRSA infection but also accelerated the wound regeneration process. Furthermore, the NFs displayed the ability to promote the M2 Mφ phenotype with enhanced collagen deposition, angiogenesis, and cell proliferation. CONCLUSION: Overall, the NFs displayed great potential as promising therapeutics for healing infected diabetic wounds through a sequential photothermal antibacterial and M2 Mφ polarization strategy.

Expression characteristics of immune factors in secondary lymphoid organs of newborn, juvenile and adult yaks (Bos grunniens).

Little is known about lymphoid organ development in yaks. In this study, we characterize and evaluate the main markers of T cell, B cell, plasma cell and antigen-presenting cell in the mesenteric lymph nodes, spleen and hemal node in newborn, juvenile and adult yaks by immunohistochemistry, real-time quantitative polymerase chain reaction and western blotting. The structures of all organs were not fully developed in newborn. The CD3+ cells were mainly located in the paracortex area of the mesenteric lymph node and the T cell dependent area in the hemal node and spleen. CD79a+ cells were mainly detected in the lymphoid follicles. The expression of CD3 and CD79a increased from newborn to juvenile and then decreased in adults. The expression of CD3 was always higher in the spleen and CD79a was higher in the mesenteric lymph node. IgG+ and IgA+ cells were observed in all examined samples, except in newborn yak hemal node. IgG and IgA were up-regulated with age and the highest expression was observed in the mesenteric lymph node. The SIRPα and CD68 were widely expressed. A significant feature was that the SIRPα expression in the spleen was lowest in newborns but highest in juvenile and adult yaks. The expression of CD68 in the hemal node was highest in all groups and increased from newborn to adult yaks. This study sheds light on the relationship between the morphology and function of these organs and provides useful references for normal yak lymphoid organ development.

TGF-ß and HSP70 profiles during transformation of yak hair follicles from the anagen to catagen stage.

Transforming growth factor-ß (TGF-ß) and heat shock protein 70 (HSP70) are important for the hair follicle (HF) cycle, but it is unclear whether they participate in HF regression in yak skin. In this study, we investigated the role of TGF-ß, TGF-ßRII, and HSP70 in the transition from anagen to catagen of HFs. The results showed that TGF-ß2 transcription was significantly higher than that of TGF-ß1 and TGF-ß3 in the same periods. Meanwhile, the expressions of TGF-ß2, TGF-ßRII, and caspase-3 were higher in the catagen phase than that in mid-anagen, and some TGF-ßRII-positive HF cells were terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive. Moreover, the HSP70 protein levels in mid-anagen were higher than those in late-anagen and catagen. These results suggested that TGF-ß2 plays a major role in catagen induction in yak HFs, which might be achieved via TGF-ßRII-mediated apoptosis in HF epithelial cells. In contrast, HSP70 might protect epithelial cells from apoptosis and ultimately inhibit HF regression. In conclusion, TGF-ß2 has positive effects, whereas HSP70 has negative effects, on catagen induction.

Yak (Bos grunniens) Tonsils: Morphological Description and Expression of IgA and IgG.

This study aimed to describe the morphology, expression of IgA and IgG in adult yak tonsils. The 12 clinically healthy yak tonsils [3- to 6-year old, n = 12] were examined for morphology using light, and transmission electron microscopes. Expression of IgA and IgG was measured by qRT-PCR, ELISA, and immunohistochemistry. The results showed that the palatine tonsil, the tonsil of the soft palate, and the lingual tonsil were oropharyngeal tonsils. The stratified squamous epithelia covering them had a thick underlying layer of connective tissue and their crypts were heavily infiltrated by lymphocytes. The pharyngeal tonsil and the tubal tonsil were nasopharyngeal tonsils. The epithelia of them was predominantly pseudostratified columnar ciliary epithelium, which were loosely arranged with a number of desmosomes or intermediate junctions variably connecting them. The expression levels of IgA and IgG mRNA and protein from high to low was in the pharyngeal tonsil, palatine tonsil, tonsil of the soft palate, lingual tonsil, and tubal tonsil, respectively. Interestingly, the expression of IgG was very significantly higher than that of IgA in yak tonsils (P < 0.01). Both the IgA and IgG ASCs were distributed in the subepithelial areas of the non-reticular crypt epithelium, especially areas of pseudostratified columnar ciliary epithelium, the reticular crypt epithelium, lymphoid follicles, interfollicular areas, and with some of the positive cells aggregating around the glands. The results indicated that the tonsils were not only typical secondary lymphoid organs but also lymphoepithelial structures. IgG could be a significant component of mucosal immune responses in yak tonsils. Anat Rec, 302:999-1009, 2019. © 2018 Wiley Periodicals, Inc.

Age-related changes in the morphology and the distribution of IgA and IgG in the pharyngeal tonsils of yaks (Bos grunniens).

To evaluate age-related changes in the morphology as well as the expression and localization of IgA and IgG in yak pharyngeal tonsils, 20 healthy yaks were divided into four age groups [newborn (1-7 days old), juvenile (5-7 months old), adult (3-6 years old) and old (7-10 years old)]. Morphologic characteristics were observed by histological techniques. The expression and localization of IgA and IgG in pharyngeal tonsils were detected by enzyme linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. The results showed that the epithelium of the pharyngeal tonsils included nonreticular epithelium with an intact basement membrane and reticular epithelium with a discontinuous basement membrane and nonepithelial cell infiltration. In newborn yaks, only primary lymphoid follicles were observed in pharyngeal tonsils. In other age groups, both primary and secondary lymphoid follicles were observed, but some of the lymphoid follicles in the old yaks were degenerated. The number of lymphoid follicles increased from the newborn to the adult group and peaked in the adult group, but the number decreased in the old group. In addition, the age-related trends of IgA and IgG protein expression were similar to those of the number of lymphoid follicles. The concentration of IgG was significantly higher than that of IgA in all age groups. Both IgA and IgG antibody secreting cells (ASCs) were distributed in the subepithelial region of the nonreticular epithelium, the reticular epithelium, the lymphoid follicles, the interfollicular areas and in between the salivary glands. The densities of IgA and IgG ASCs in pharyngeal tonsils were similar to the expression trend of both proteins in each age group. The results indicate that the morphology and amount of lymphoid follicles in yak pharyngeal tonsils vary with age. Pharyngeal tonsils produce more IgG than IgA, indicating that IgG could be significant component of mucosal immune responses in yaks.